EP1413630B1 - Analyse de cibles biologiques utilisant une biopuce comportant un marqueur fluorescent - Google Patents

Analyse de cibles biologiques utilisant une biopuce comportant un marqueur fluorescent Download PDF

Info

Publication number
EP1413630B1
EP1413630B1 EP03104367A EP03104367A EP1413630B1 EP 1413630 B1 EP1413630 B1 EP 1413630B1 EP 03104367 A EP03104367 A EP 03104367A EP 03104367 A EP03104367 A EP 03104367A EP 1413630 B1 EP1413630 B1 EP 1413630B1
Authority
EP
European Patent Office
Prior art keywords
fluorescence
seq
oligonucleotide
flavin
oligonucleotides
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Lifetime
Application number
EP03104367A
Other languages
German (de)
English (en)
French (fr)
Other versions
EP1413630A1 (fr
Inventor
Marc Cuzin
Philippe Peltie
Marc Fontecave
Jean-Luc Decout
Cécile DUEYMES
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Universite Joseph Fourier Grenoble 1
Commissariat a lEnergie Atomique et aux Energies Alternatives CEA
Original Assignee
Commissariat a lEnergie Atomique CEA
Universite Joseph Fourier Grenoble 1
Commissariat a lEnergie Atomique et aux Energies Alternatives CEA
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Commissariat a lEnergie Atomique CEA, Universite Joseph Fourier Grenoble 1, Commissariat a lEnergie Atomique et aux Energies Alternatives CEA filed Critical Commissariat a lEnergie Atomique CEA
Publication of EP1413630A1 publication Critical patent/EP1413630A1/fr
Application granted granted Critical
Publication of EP1413630B1 publication Critical patent/EP1413630B1/fr
Anticipated expiration legal-status Critical
Expired - Lifetime legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00497—Features relating to the solid phase supports
    • B01J2219/00527—Sheets
    • B01J2219/00529—DNA chips
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/0054—Means for coding or tagging the apparatus or the reagents
    • B01J2219/00572—Chemical means
    • B01J2219/00576—Chemical means fluorophore
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00608—DNA chips
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/0068—Means for controlling the apparatus of the process
    • B01J2219/00693—Means for quality control
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/0068—Means for controlling the apparatus of the process
    • B01J2219/00702—Processes involving means for analysing and characterising the products
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00722—Nucleotides
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof

Definitions

  • the present description relates to an analysis support or "biochip” for the determination of biological targets of the DNA or RNA type.
  • This analysis support has applications in many fields, in particular in biology for genome sequencing, search for mutations, development of new drugs, etc.
  • An analysis support of this type comprises a plurality of oligonucleotide probes capable of giving rise to hybridization with the biological targets to be analyzed.
  • the hybridization corresponds to the pairing of the target strands with the complementary DNA strands arranged on the support. To determine the nature of the targets, it is therefore necessary to be able to locate which sites of the support and therefore which oligonucleotides have given rise to hybridization.
  • the hybridization of the biological targets on the support is determined by means of a fluorescent marker which is associated with the biological targets, after extraction of the zones of interest (lysis and possible amplification).
  • the sites where the hybridization has taken place are determined by carrying out an excitation of all the fluorescent markers, followed by a reading of the sites to detect the fluorescence light re-emitted by the markers.
  • the sites for which a fluorescence light is detected are those which have fixed target molecules. Reading systems adapted to various biochips are described for example in US-A-5,578,832 [2] and US-A-5,646,411 [3].
  • This technique has the drawback of requiring the labeling of the biological target before it comes into contact with the analysis support, which poses certain practical problems as regards the quantification and / or the yield of the marking and the delay for the 'carry out.
  • marking at a given stage freezes the situation and prevents subsequent dynamic measurements that can follow a hybridization reaction.
  • the patent US 5843 655 discloses a method of controlling a support using oligonucleotides labeled with a fluorescent probe to locate and determine the amount of oligonucleotides attached to said support.
  • the present description has for object precisely a support of analysis, or biopuce, which makes it possible to ensure under better conditions the prior control of manufacture, and then to facilitate and make more sensitive the determination of the sites of the support on which produces a hybridization.
  • the analysis support comprises a plurality of oligonucleotides attached to this support, and it is characterized in that each of said oligonucleotides is labeled with a fluorescent compound such that the fluorescence of an oligonucleotide labeled with this compound is attenuated. after hybridization of this labeled oligonucleotide with a complementary oligonucleotide.
  • the fluorescence attenuation can consist of a variation of the fluorescence intensity, a variation of the time constant of fluorescence, or a spectral shift in wavelength of fluorescence.
  • a fluorescent compound having a fluorescence intensity attenuated by hybridization is used.
  • this attenuation represents 30 to 99% of the fluorescence intensity before hybridization
  • Fluorescent compounds having these characteristics are constituted for example by flavin, deazaflavin and their derivatives. Such compounds are attached to oligonucleotides via a spacer arm, for example of the - (CH 2 ) n - type, to give flavin (deazaflavin) -oligonucleotide conjugates.
  • a synthesis of conjugates of this type is described by C. Frier et al in J. Org. Chem., 62, 1997, pages 3520-3528 [4].
  • This synthesis consists in fixing a flavin derivative on the oligonucleotide by a phosphoramidite or H-phosphonate coupling method starting from a derivative of formula: with n being an integer from 2 to 8, for example 6.
  • the synthesis strategy makes it possible to introduce the flavinic group, including in automated oligonucleotide synthesis methods. This synthesis can also be used for the manufacture of oligonucleotide-deazaflavine conjugates.
  • the oligonucleotide is labeled with a group of formula - (CH 2 ) n -R 1 in which n is an integer ranging from 2 to 8, and R 1 represents a group corresponding to one of the following formulas
  • flavin, deazaflavin or their derivatives as a fluorescent compound in the invention is particularly interesting.
  • flavins are molecules with an intense yellow-green fluorescence, very characteristic.
  • the fluorescence excitation spectrum is identical to the visible light absorption spectrum, with 2 maxima at 374 and 446 nanometers, and their fluorescence emission spectrum consists of a wide band with a maximum around 520 nanometers.
  • Deazaflavins absorb at 320 and 396 nanometers and have higher fluorescence intensities (emission at 452 nanometers) than flavins.
  • the description also relates to a method for analyzing biological targets by contacting these targets with an analysis support comprising a plurality of oligonucleotides, and determining hybridization (s) between the targets. and the oligonucleotides of the support, characterized in that each of the oligonucleotides of the support is labeled with a fluorescent compound which exhibits a variation of fluorescence during the hybridization of the oligonucleotide labeled with the oligonucleotide complementary, and in that one determines the oligonucleotides of the support having given rise to hybridization by measuring the fluorescence corresponding to each oligonucleotide of the support before and after putting the contact in contact with the targets.
  • the targets are further labeled by a second fluorescent compound whose emission wavelength is different from that of the fluorescent compound of the oligonucleotides, or first fluorescent compound, and the oligonucleotides having gave rise to a fluorescence variation by measuring the fluorescence of this second compound.
  • the fluorescent compound of the oligonucleotides of the support, or first fluorescent compound is chosen from flavin, deazaflavin and their derivatives
  • the second fluorescent compound is chosen from stable fluorophores used on DNA chips and emitting preferentially in the visible (Rhodamine, CY 3 , CY 5 etc.), for example redshifted with respect to flavin.
  • the oligonucleotides of which are labeled with a fluorescent compound, it is possible moreover to control the quality of the support.
  • the subject of the invention is therefore a method for controlling an analysis medium comprising a plurality of oligonucleotides attached to said support, characterized in that the fixed oligonucleotides are oligonucleotides labeled with a fluorescent compound and in that the quantity of oligonucleotides fixed on the support is localized and determined by measuring the fluorescence of the fluorescent compound, each of said oligonucleotides being labeled with a group of formula - (CH 2 ) m -R 1 as defined above.
  • a flavine-T 16 conjugate (SEQ ID NO: 9) of the formula
  • 0.1 to 10 ⁇ mol.L -1 of the T 11 -flavin conjugate is brought into contact with 1 equivalent of the oligonucleotide (SEQ ID NO: 1), in a 10 mM Tris-HCl buffer, pH 7. With 0.1 mM-1MNaCl.
  • the excitation is carried out at 460 nanometers and the fluorescence emission is measured around 540 nanometers.
  • the fluorescence emission only represents 5% of the measured emission on the conjugate T 11 -flavine alone.
  • the fluorescence emission represents 6% of the fluorescence emission of the conjugate alone.
  • the presence of the target (SEQ ID NO: 2) of the oligonucleotide T 16 leads to a fluorescence quenching of flavin due to the hybridization between the target and the oligonucleotide T 16 .
  • the properties of the flavin-conjugated HIV oligonucleotide (SEQ ID NO: 10) are tested in the presence of the HIV duplex formed by SEQ ID NO: 6 and SEQ ID NO: 7.
  • the fluorescence emission represents 90% of the initial emission. There was no hybridization between the F1-HIV conjugate and the duplex since the HIV target had already given rise to hybridization.
  • the figure 1 illustrates the results obtained, namely the evolution of the fluorescence intensity at 452 nanometers as a function of the number of equivalents of the poly A target (SEQ ID NO: 1).
  • the figure 2 illustrates the results obtained, namely the fluorescence intensity at 520 nanometers as a function of the number of equivalents of the poly A target (SEQ ID NO: 1).
  • the stability of the T 11 -flavine complex (SEQ ID NO: 8) is studied with the complementary SEQ ID NO: 1.
  • Gel-retardation experiments and measurement of the melting temperature Tm are carried out. It has thus been possible to show that the presence of a flavin or of a deazaflavin at the end of an oligonucleotide confers an important additional stability of the complex makes this oligonucleotide with the complementary oligonucleotide.
  • the T 11 sequence leads to a complex with the oligonucleotide SEQ ID NO: 1 with a Tm of 23 ° C (37 ° C to 1 M NaCl), while the conjugate T 11 -flavine (SEQ ID NO: 8) gives, with SEQ ID NO: 1, a complex with a Tm of 28 ° C (42 ° C to 0.1 M NaCl).
  • a conjugate of a 20 mer oligonucleotide and flavin (SEQ ID NO: 12) is contacted: with the complementary oligonucleotide (SEQ ID NO: 13): ctagcctgat gaaaggggaa gtggtggggg agacatagcc in increasing concentration, under the same conditions as those of Examples 2 to 4, and the fluorescence spectrum is measured around 520 nanometers at the spectrofluorimeter.
  • the excitation wavelength is 446 nanometers and the maximum emission corresponding to flavin is 520 nanometers.
  • the measurement is made instantaneously, then 5 minutes later, with each time a higher concentration of the complementary target (SEQ ID NO: 13).
  • the figure 3 illustrates the fluorescence spectra obtained without the complementary oligonucleotide, then with 0.2, 0.4, 0.6, 0.8, 1, 1.2 and 1.4 equivalents of the complementary oligonucleotide SEQ ID NO: 13 .
  • the quenching can correspond to various processes that can be either energy transfer, collisional quenching, or complex quenching that corresponds to probe-target hybridization. .
  • the figure 4 illustrates the evolution of I 0 / I and of ⁇ 0 / ⁇ , as a function of [Q]. As the temperature increases, the slope of the right increases.
  • the figure 5 illustrates the evolution of I 0 / I as a function of [Q].
  • ⁇ 0 / ⁇ 1 and the slope decreases when the temperature increases, which is normal since an increase in temperature causes a partial denaturation of the complex, so the fluorophore which was extinguished is released.
  • the figure 8 illustrates the variation of K app as a function of the concentration [Q] in equivalents from the results of the figure 3 .
  • the target is attached to a second fluorophore, not subject to "quenching" and strongly shifted in fluorescence wavelength relative to the fluorescent compound.
  • this fluorophore can be excited in parallel and will reveal the effective presence of the hybridization complex.
  • a fluorochrome such as CY 3 which has an excitation wavelength equal to 543.5 nanometers and an equal emission wavelength. at 580 nanometers. It can be revealed with a scanner using a low power green HeNe laser (1 mW) as it is done in a microchip reading system.
  • This double labeling that is to say that of the oligonucleotide attached to the biochip by means of a first fluorescent compound such as flavin, and that of the targets to be determined by a second fluorescent compound such as CY 3 , has undeniable advantages over a simple traditional marking.
  • the presence of flavin allows a control of the quality of the probes before hybridization, making a total image of the chip so that all sites have fluorescent probes. It is then possible to map the defects of the chip, (non-homogeneous image in fluorescence intensity); there is also possibility to take advantage of the redundancy by putting the same type of probe on several contiguous sites.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Analytical Chemistry (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Optical Measuring Cells (AREA)
EP03104367A 2000-02-23 2001-02-22 Analyse de cibles biologiques utilisant une biopuce comportant un marqueur fluorescent Expired - Lifetime EP1413630B1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
FR0002236 2000-02-23
FR0002236A FR2805348B1 (fr) 2000-02-23 2000-02-23 Analyse de cibles biologiques utilisant une biopuce comportant un marqueur fluorescent
EP01907873A EP1266030B1 (fr) 2000-02-23 2001-02-22 Analyse de cibles biologiques utilisant une biopuce comportant un marqueur fluorescent

Related Parent Applications (2)

Application Number Title Priority Date Filing Date
EP01907873A Division EP1266030B1 (fr) 2000-02-23 2001-02-22 Analyse de cibles biologiques utilisant une biopuce comportant un marqueur fluorescent
EP01907873.2 Division 2001-02-22

Publications (2)

Publication Number Publication Date
EP1413630A1 EP1413630A1 (fr) 2004-04-28
EP1413630B1 true EP1413630B1 (fr) 2012-01-11

Family

ID=8847290

Family Applications (2)

Application Number Title Priority Date Filing Date
EP03104367A Expired - Lifetime EP1413630B1 (fr) 2000-02-23 2001-02-22 Analyse de cibles biologiques utilisant une biopuce comportant un marqueur fluorescent
EP01907873A Expired - Lifetime EP1266030B1 (fr) 2000-02-23 2001-02-22 Analyse de cibles biologiques utilisant une biopuce comportant un marqueur fluorescent

Family Applications After (1)

Application Number Title Priority Date Filing Date
EP01907873A Expired - Lifetime EP1266030B1 (fr) 2000-02-23 2001-02-22 Analyse de cibles biologiques utilisant une biopuce comportant un marqueur fluorescent

Country Status (9)

Country Link
US (1) US6861515B2 (ja)
EP (2) EP1413630B1 (ja)
JP (1) JP4729226B2 (ja)
AT (2) ATE275641T1 (ja)
AU (1) AU2001235742A1 (ja)
CA (1) CA2399258A1 (ja)
DE (1) DE60105399T2 (ja)
FR (1) FR2805348B1 (ja)
WO (1) WO2001063282A2 (ja)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001073118A2 (en) * 2000-03-29 2001-10-04 Lgc (Teddington) Limited Hybridisation beacon and method of rapid sequence detection and discrimination
JP2004527746A (ja) * 2001-03-16 2004-09-09 キューティエル・バイオシステムズ・リミテッド・ライアビリティ・カンパニー 蛍光ポリマー超消光に基づくバイオアッセイ
FR2827304B1 (fr) 2001-07-16 2004-05-21 Commissariat Energie Atomique Procede et support d'analyse biologique utilisant des oligonucleotides comportant un marqueur activable enzymatiquement
DE10155055A1 (de) * 2001-11-09 2003-05-28 Friz Biochem Gmbh Fluoreszenz-Quenchen zur Detektion von Nukleinsäure-Oligomer-Hybridisierungsereignissen bei hohen Salz-Konzentrationen
CN1424405A (zh) * 2001-12-14 2003-06-18 宋克 基因芯片分子探针及相关技术

Family Cites Families (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5925525A (en) * 1989-06-07 1999-07-20 Affymetrix, Inc. Method of identifying nucleotide differences
US5538848A (en) * 1994-11-16 1996-07-23 Applied Biosystems Division, Perkin-Elmer Corp. Method for detecting nucleic acid amplification using self-quenching fluorescence probe
US5578832A (en) 1994-09-02 1996-11-26 Affymetrix, Inc. Method and apparatus for imaging a sample on a device
US5843655A (en) * 1995-09-18 1998-12-01 Affymetrix, Inc. Methods for testing oligonucleotide arrays
US5861247A (en) * 1996-01-26 1999-01-19 University Of Chicago Rapid method to detect duplex formation in sequencing by hybridization methods
US5646411A (en) 1996-02-01 1997-07-08 Molecular Dynamics, Inc. Fluorescence imaging system compatible with macro and micro scanning objectives
SE522077C2 (sv) * 1997-09-05 2004-01-13 Lightup Technologies Ab Förfarande att välja sekvens hos sond för nukleinsyrahybridisering
AU742599B2 (en) * 1997-07-22 2002-01-10 Qiagen Genomics, Inc. Multiple functionalities within an array element and uses thereof
WO1999060158A1 (fr) * 1998-05-19 1999-11-25 Laboratory Of Molecular Biophotonics Phase solide utilisee pour detecter de l'acide nucleique et procede de detection d'acide nucleique
WO2001038585A2 (en) * 1999-11-24 2001-05-31 The Regents Of The University Of California Polymer arrays and methods of using labeled probe molecules to identify and quantify target molecule expression

Also Published As

Publication number Publication date
US20030165914A1 (en) 2003-09-04
EP1266030B1 (fr) 2004-09-08
DE60105399T2 (de) 2005-09-15
CA2399258A1 (fr) 2001-08-30
FR2805348B1 (fr) 2002-07-12
ATE541050T1 (de) 2012-01-15
JP2003529751A (ja) 2003-10-07
AU2001235742A1 (en) 2001-09-03
WO2001063282A3 (fr) 2002-04-04
DE60105399D1 (de) 2004-10-14
WO2001063282A2 (fr) 2001-08-30
US6861515B2 (en) 2005-03-01
EP1266030A2 (fr) 2002-12-18
FR2805348A1 (fr) 2001-08-24
EP1413630A1 (fr) 2004-04-28
JP4729226B2 (ja) 2011-07-20
ATE275641T1 (de) 2004-09-15

Similar Documents

Publication Publication Date Title
US11384391B2 (en) Flourescene energy transfer-based single molecule/ensemble DNA sequencing by synthesis
US20110111975A1 (en) Probe for nucleic acid sequencing and methods of use
US20070059693A1 (en) Hybridization-based biosensor containing hairpin probes and use thereof
WO2004059302A1 (fr) Lecteur de puces de type biopuces, et procedes associes
KR102425463B1 (ko) 핵산 서열분석을 위한 표지된 뉴클레오티드 조성물 및 방법
Trabesinger et al. Detection of individual oligonucleotide pairing by single-molecule microscopy
EP1413630B1 (fr) Analyse de cibles biologiques utilisant une biopuce comportant un marqueur fluorescent
US20020068282A1 (en) Detection/quantification of targeted nucleotide chains, and detection/quantification of multi-stranded nucleotide chains by fluorescence
EP1706509B1 (fr) Puce d'analyse avec gamme etalon, trousses et procedes d'analyse
EP1121461B1 (fr) Sondes fluorescentes de peinture chromosomique
CN112630198A (zh) 一种基于回音壁模式光学微腔的传感检测方法
EP1344062B1 (fr) Support pour determiner un analyte comportant un filtre spectral selectif
US20220364167A1 (en) Flourescene energy transfer-based single molecule/ensemble dna sequencing by synthesis
EP0882233B1 (fr) Procede et dispositif de traitement par complexation d'un milieu liquide
JPH11164700A (ja) Ｄｎａ検出方法
FR3125824A1 (fr) Dispositif et procédé de détection multiplexée de séquences d’acides nucléiques
EP3137879B1 (fr) Dispositif et procede d'analyse d'analyte(s) d'un fluide
FR2827304A1 (fr) Procede et support d'analyse biologique utilisant des oligonucleotides comportant un marqueur activable enzymatiquement
Smith et al. Detection of DNA and P-450s on silver colloidal nanoparticles by surface-enhanced resonance Raman scattering (SERRS)
FR2836926A1 (fr) Substrat oligonucleotidique et procede permettant d'analyser la presence d'activites de reparation de lesions de l'adn
FR2822234A1 (fr) Procede de determination locale de parametres structuraux de molecules, notamment biochimiques ou pharmacologiques et utilisations dudit procede
JP2001033440A (ja) 乾式蛍光測定による多重鎖核酸の検出／定量方法

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

AC Divisional application: reference to earlier application

Ref document number: 1266030

Country of ref document: EP

Kind code of ref document: P

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE TR

RTI1 Title (correction)

Free format text: ANALYSIS OF BIOLOGICAL TARGETS USING A BIOCHIP COMPRISING A FLUORESCENT MARKER

17P Request for examination filed

Effective date: 20041022

AKX Designation fees paid

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE TR

RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: UNIVERSITE JOSEPH FOURIER DE GRENOBLE

Owner name: COMMISSARIAT A L'ENERGIE ATOMIQUE

17Q First examination report despatched

Effective date: 20080226

RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: UNIVERSITE JOSEPH FOURIER DE GRENOBLE

Owner name: COMMISSARIAT A L'ENERGIE ATOMIQUE ET AUX ENERGIES

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: UNIVERSITE JOSEPH FOURIER DE GRENOBLE

Owner name: COMMISSARIAT A L'ENERGIE ATOMIQUE ET AUX ENERGIES

GRAS Grant fee paid

Free format text: ORIGINAL CODE: EPIDOSNIGR3

GRAA (expected) grant

Free format text: ORIGINAL CODE: 0009210

AC Divisional application: reference to earlier application

Ref document number: 1266030

Country of ref document: EP

Kind code of ref document: P

AK Designated contracting states

Kind code of ref document: B1

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE TR

REG Reference to a national code

Ref country code: GB

Ref legal event code: FG4D

Free format text: NOT ENGLISH

REG Reference to a national code

Ref country code: CH

Ref legal event code: EP

REG Reference to a national code

Ref country code: AT

Ref legal event code: REF

Ref document number: 541050

Country of ref document: AT

Kind code of ref document: T

Effective date: 20120115

REG Reference to a national code

Ref country code: IE

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: DE

Ref legal event code: R096

Ref document number: 60145960

Country of ref document: DE

Effective date: 20120315

REG Reference to a national code

Ref country code: NL

Ref legal event code: VDEP

Effective date: 20120111

REG Reference to a national code

Ref country code: FR

Ref legal event code: TP

Owner name: COMMISSARIAT A L'ENERGIE ATOMIQUE ET AUX ENERG, FR

Effective date: 20120515

REG Reference to a national code

Ref country code: GB

Ref legal event code: 732E

Free format text: REGISTERED BETWEEN 20120621 AND 20120627

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: NL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20120111

REG Reference to a national code

Ref country code: IE

Ref legal event code: FD4D

REG Reference to a national code

Ref country code: DE

Ref legal event code: R081

Ref document number: 60145960

Country of ref document: DE

Owner name: COMMISSARIAT A L'ENERGIE ATOMIQUE ET AUX ENERG, FR

Free format text: FORMER OWNER: COMMISSARIAT A L'ENERGIE ATOMIQ, UNIVERSITE JOSEPH FOURIER DE GR, , FR

Effective date: 20120608

Ref country code: DE

Ref legal event code: R081

Ref document number: 60145960

Country of ref document: DE

Owner name: COMMISSARIAT A L'ENERGIE ATOMIQUE ET AUX ENERG, FR

Free format text: FORMER OWNER: COMMISSARIAT A L'ENERGIE ATOMIQ, UNIVERSITE JOSEPH FOURIER DE GR, , FR

Effective date: 20120120

Ref country code: DE

Ref legal event code: R081

Ref document number: 60145960

Country of ref document: DE

Owner name: COMMISSARIAT A L'ENERGIE ATOMIQUE ET AUX ENERG, FR

Free format text: FORMER OWNERS: COMMISSARIAT A L'ENERGIE ATOMIQUE ET AUX ENERGIES ALTERNATIVES, PARIS, FR; UNIVERSITE JOSEPH FOURIER DE GRENOBLE, GRENOBLE, FR

Effective date: 20120608

Ref country code: DE

Ref legal event code: R081

Ref document number: 60145960

Country of ref document: DE

Owner name: COMMISSARIAT A L'ENERGIE ATOMIQUE ET AUX ENERG, FR

Free format text: FORMER OWNERS: COMMISSARIAT A L'ENERGIE ATOMIQUE, PARIS, FR; UNIVERSITE JOSEPH FOURIER DE GRENOBLE, GRENOBLE, FR

Effective date: 20120120

BERE Be: lapsed

Owner name: UNIVERSITE JOSEPH FOURIER DE GRENOBLE

Effective date: 20120228

Owner name: COMMISSARIAT A L'ENERGIE ATOMIQUE ET AUX ENERGIES

Effective date: 20120228

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: FI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20120111

Ref country code: PT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20120511

Ref country code: GR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20120412

REG Reference to a national code

Ref country code: AT

Ref legal event code: MK05

Ref document number: 541050

Country of ref document: AT

Kind code of ref document: T

Effective date: 20120111

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MC

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20120229

Ref country code: CY

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20120111

REG Reference to a national code

Ref country code: CH

Ref legal event code: PL

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: CH

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20120229

Ref country code: SE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20120111

Ref country code: IE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20120111

Ref country code: LI

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20120229

Ref country code: DK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20120111

PLBE No opposition filed within time limit

Free format text: ORIGINAL CODE: 0009261

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20120111

26N No opposition filed

Effective date: 20121012

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: BE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20120228

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: AT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20120111

REG Reference to a national code

Ref country code: DE

Ref legal event code: R097

Ref document number: 60145960

Country of ref document: DE

Effective date: 20121012

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: ES

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20120422

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: TR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20120111

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: DE

Payment date: 20140211

Year of fee payment: 14

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LU

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20120222

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: FR

Payment date: 20140228

Year of fee payment: 14

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: GB

Payment date: 20140219

Year of fee payment: 14

REG Reference to a national code

Ref country code: DE

Ref legal event code: R119

Ref document number: 60145960

Country of ref document: DE

GBPC Gb: european patent ceased through non-payment of renewal fee

Effective date: 20150222

REG Reference to a national code

Ref country code: FR

Ref legal event code: ST

Effective date: 20151030

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: GB

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20150222

Ref country code: DE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20150901

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: FR

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20150302